Columns used in liquid chromatography typically comprise a tubular body enclosing a porous chromatography medium through which a carrier liquid flows, with separation taking place by material collection between the carrier liquid and solid phase of the porous medium. Prior to any separation process, the bed has to be prepared starting from the slurry of particles that has to be introduced into the column. The process of bed formation is called ‘the packing procedure’ and a correctly packed bed is a critical factor influencing the performance of a column containing a packed bed. The goal of the packing procedure is to provide a bed compressed by the optimum amount of compression—the optimum compression factor.
In detail, the porous medium is formed by consolidating a suspension of discrete particles, known as “slurry” that is pumped or poured or sucked into the column, usually from one end. Consolidation of the slurry into a packed bed is typically achieved by filtering it against a particle retaining filter and further compressing the formed filter cake so that it is packed into a volume which is less than the volume that it would have occupied if it had sedimented under the influence of only gravity to form a sedimented bed. The degree of compression depends upon the type of chromatography medium and typically ranges from in between 2-20%. The efficiency of subsequent chromatographic separations relies on the liquid distribution and collection system at the fluid inlet and outlet of the packed bed, but primarily on the homogeneity and stability of the packed bed formed. If the packed bed is not homogeneous and stable a deleterious effect will be experienced for chromatographic separations performed on the bed. Homogeneity and stability of the packed bed depend upon the optimum degree of compression, which must be determined experimentally for each column size (width or diameter), bed height and bed medium.
Several methods are known in the art for packing columns (see, for example, US 2003/0146159). “Flow packing” is a method typically utilised for the preparation of analytical columns (i.e. columns of about 1-10 mm column diameter) and semi-preparative columns (i.e. columns of 10-100 mm column diameter) or even larger. In flow packing, one end of a column is closed by a frit or a filter. At the other end, a slurry or suspension of the packing material is pumped or poured into the column tube. A filtration bed builds up against the frit and grows until a filter cake has formed. The bed is then compressed further to its “target bed height” by percolating a number of column volumes (ca. 3-10) of a packing solvent at a flow rate that is higher than the flow rates used in operation. Consolidation and subsequent compression take place under the influence of the seepage force, that is the reaction of the bed to the pressure gradient required to maintain the flow rate of the stream of liquid percolating through the bed. Once the bed is compressed by the flow, the flow is stopped, the outlet at the bottom of the column is closed and an adapter or upper end cell is adjusted to the target height of the compressed bed. This adjustment is done quickly to avoid a relaxation of the compressed bed exceeding the target bed height.
The flow packing method has the disadvantage that beds of packing material compressed in this manner are axially heterogeneous during the flow compression step yielding highest compression close to the outlet of the column and zero compression at the top of the packed bed. This results in a major relaxation of the bed and a possible re-arrangement of the particles once the packing flow has been stopped and the upper end cell has been brought into position. The gradient in bed compression inherent to this method may result in poor bed stability and poor column efficiency depending on the type of medium and the packed bed geometry.
Standard methods of flow packing may not be suitable for the wide bore columns used in preparative chromatography. Among other factors, it is often undesirable to design equipment that requires application of a packing flow rate and thus a packing pressure substantially higher than the pressure required for subsequent operation. To remedy this problem, packing methods relying on mechanical axial compression are used. Axial compression methods achieve the bed compression by an axial movement of the adapter (end cell). Hereby, the need for high liquid pressure in the column space during packing is removed. A further advantage of the axial compression method is that the bed is compressed homogeneously in axial direction, which avoids the problems of relaxation and particle re-arrangements that occur with the flow packing method.
Radial gradients in bed compression and bed voidage occur with both methods described, which is due to wall friction effects. The impact of the radial heterogeneity depends on the bed geometry, i.e. the ratio of diameter to height. As described above, it is the gradient of compression and bed voidage in axial direction that is substantially different between the flow packing method and the axial compression method.
A disadvantage of axial compression is that columns packed using this method require a means for moving the end cell and a means for controlling this movement. Typical methods for the movement are motor drives or hydraulic systems. As these are attached to or built in to the column, the cost and mechanical complexity of axial compression columns is substantially higher than for flow packing columns.
Prior to any consolidation and compression the medium has to be introduced into the column. Large scale columns are preferably prepared by injecting slurry through a central slurry nozzle or valve into the column. This enables a closed system approach which is preferable for sanitary reasons. Columns based on a slurry valve design may be designed for axial compression packing using a movable adapter or for flow packing using a fixed end cell.